The present invention is directed to the production of cellulose-degrading enzymes in genetically recombinant plants and the recombinant plants themselves.
Complete bibliographic citations for the non-patent references discussed hereinbelow are included in the Bibliography section, immediately preceding the claims. All of the references cited below are incorporated herein by reference.
Lignocellulosic plant matter, such as agricultural and forestry waste, as well as energy crops produced specifically for biomass, offer tremendous potential for the renewable production of fuel and as chemical feedstocks. However, production cost for desired products such as alcohols from lignocellulosic material is significantly higher than the production cost of equivalent alternatives. However, the prospect, either real or perceived, of limited fossil fuel reserves, along with the geo-political issues which swirl about petroleum-producing countries and regions, renders the production of basic chemical feedstocks and fuels from local, renewable sources an attractive alternative to fossil fuels.
For instance, alcohols have the potential to be excellent alternative transportation fuels if their production costs can be lowered. Brazil has sponsored several programs to replace car engines which run on gasoline alone to engines which run on ethanol or a gasoline-ethanol mix.
Unfortunately, the production of ethanol and other feedstock chemicals from lignocellulosic material is far more complex than an analogous production utilizing a starch-based starting material. Compared to lignocellulosic materials, starch is a simple polymer which is readily hydrolyzed to glucose. Yeasts can then be used to convert the glucose to ethanol.
In contrast, lignocellulosic biomass is a much more complex substrate in which crystalline cellulose is embedded within a matrix of hemicellulose and lignin. The intricate structure and relative inaccessibility of these substrates requires pre-treatment for the disruption of the lignocellulosic material, as well as hydrolysis of hemicellulose and lignin into xylose and phenolic compounds, respectively. (See, for instance, Micelli et al. (1996), Belkacemi et al. (1996), and Grohmann et al. (1992).)
Several enzymes which degrade lignocellulosic material, commonly referred to as xe2x80x9ccellulases,xe2x80x9d are known. The term xe2x80x9ccellulasexe2x80x9d shall be used herein to refer to any and all enzymes which catalyze the cleavage of cellulosic or lignocellulosic materials. Explicitly, but not exclusively, included within this definition are those cellulases which fall under the Enzyme Classification heading EC 3.2.1.x. Various genes encoding cellulases have also been isolated and characterized.
For instance, genes which encode endoglucanases from the fungus Trichoderma reesei are known and have been successfully incorporated and expressed in yeast. See, for instance, Pentilla et al. (1987). Likewise, cellulase E2 (EC 3.2.1.4) and cellulase E3 (EC 3.2.1.91) from the thermo-tolerant bacterium Thermomonospora fusca are known. See Lao et al. (1991), Spezio et al. (1993) and Zhang et al. (1995).
From a functional viewpoint, cellulases are catagorized into two large sub-groups based upon whether they catalyze cleavage from the cellulose chain ends (exocellulases) or if they catalyze cleavage in the middle of the cellulose chain (endocellulases). For instance, cellobiohydrolase I of T. reesei (CBH I, EC 3.2.1.91) is an exocellulase, which degrades crystalline cellulose by cleavage from the chain ends. By way of further illustration, CBH I is a 68 kDa protein with a two-domain architecture which is shared by many cellulases. In this chemical architecture, a large catalytic domain is joined to a cellulose-binding domain (CBD) through a flexible linker region. See Divne et al. (1994). Similarly, cellulase E3 of T. fusca is also an exocellulase.
Different types of cellulases exhibit synergistic activity on complex substrates. This synergism, especially between exocellulases, is believed to be due to differences in their patterns of absorption to and hydrolysis of complex cellulose substrates. See Henrissat et al. (1995).
Illustratively, cellulase E2 of T. fusca is a 40 kDa endocellulase which cleaves the cellulose chain internally. Such cleavage generates more chain ends for attack by exocellulases. Consequently when CBH I, E2, and E3 cellulases are combined, their activity together is approximately 5-fold greater than their additive individual activities. (See, for instance, Irwin et al. (1993) and WO 94/26880.) It is important to note that proteolytic fragments of cellulases can substitute for the intact enzymes in synergistic mixtures. For example, when combined with T. fusca E3 and CBH I, the catalytic domain of T. fusca E2 (xe2x80x9cE2cdxe2x80x9d) is as active as the intact enzyme in the digestion of filter paper substrate, Irwin et al. (1993).
A wide range of compositions containing cellulases are described in the patent literature. For instance, Evans et al., U.S. Pat. No. 5,432,074, describe the use of a formulation consisting essentially of a combination of xylanase and xylosidase, but being essentially free of glucanase and cellobiohydrolase. The formulation also contains a lactic acid-producing bacteria. The formulation is used to treat silage to increase its nutritive value. In operation, the action of the xylanase and xylosidase enzymes degrades non-cellulosic polysaccharides found in the silage material thereby producing sugars for fermentation.
Heterodimers of different types of cellulose-degrading enzymes are described in WO 94/29460. Here, a xcex2-glucosidase molecule and a cellobiohydrolase molecule (i.e., an exocellulase) are chemically bonded to one another by a crosslinking reagent to yield a single molecule which retains the enzymatic activities of the two separate molecules.
Expression constructs which contain cellulase genes for the transformation of yeast have been constructed. For example, Knowles et al., U.S. Pat. No. 5,529,919, describe the transformation of S. cerevisiae to contain and express a thermostable xcex2-endoglucanase (EG I) of T. reesei. 
Likewise, attempts have been made to produce transgenic plants which express cellulose-degrading enzymes. Aspegren et al. (1995) describe transgenic suspension-cultured barley cells which express EG I of T. reesei. The cells were transformed by particle bombardment and transformed cells selected by a co-transformed antibiotic resistance marker. However, no attempt was made to regenerate complete plants from the cultured cells. Of particular note, this reference states that the production of xcex2-glucanases in plant cells may be hampered by the fact that these enzymes catalyze the hydrolysis of essential cell wall components. Attempts by these authors to stably transform tobacco cells with the same construct used to successfully transform the suspended barley cells failed. Here, the authors observed that after transient expression in tobacco protoplasts, cell wall synthesis never resumed.
The present invention is drawn to genetically recombinant plants which contain one or more exogenous gene sequences which encode one or more cellulose-degrading gene products. The gene product or products are expressed in recoverable quantities in the recombinant plants and can be isolated from the plants, if desired. In the preferred embodiment, the genetically recombinant plant expresses the gene product constituitively.
However, the invention also encompasses recombinant plants which express the gene product stage-specifically or tissue-specifically. For example, the gene product or products can be expressed in a plant tissue such as the seeds, fruit, leaves, or tubers of the transformed plant host.
The invention is further drawn to recombinant plants as noted above, wherein the plant contains two exogenous genes whose respective gene products are expressed independently of one another. This allows for different types of cellulases to be expressed in different locations within the same recombinant plant. For example, the plant host can be transformed to express two or more heterologous cellulases in different sub-cellular compartments such as the plastid, cytosol, endoplasmic reticulum, mitochondrion, inclusion body, or vacuole. In addition, chloroplast targeting can also be accomplished through the use of direct chloroplast transformation, an approach that circumvents many of the problems associated with expression of heterologous genes in the nuclear genome. Carrer et al. (1993), McBride et al. (1994).
The invention is further drawn to a method for producing cellulose-degrading enzymes. The method comprises transforming a plant host with one or more exogenous genes which encode one or more cellulose-degrading gene products such that the gene product or products are expressed in recoverable quantities. The plant matter containing the expressed protein can be used directly as a feedstock for biomass conversion, or, if desired, the exogenous enzymes so produced can be isolated and purified.
The cellulases produced by the transgenic plants of the present invention can be utilized in the same manner as conventionally-derived cellulases. For instance, cellulases produced by the transgenic plants of the present invention can be isolated and used in fermentation processes such as brewing and wine-making. Here, the cellulases function to hydrolyze cellulose and xcex2-glucans during fermentation. Or, as described in Example 4, below, whole plants transformed to express cellulases can be used directly or added to ensiled plant matter to increase the extent of fermentation of the ensiled matter. Plants transformed to express functional cellulases may also be fed directly to livestock, where the cellulase activity aids in the digestion of lignocellulosic substrates.
Cellulases produced in the transgenic plants of the present invention can also be utilized in the production of ethanol and other feedstock chemicals from lignocellulosic substrates.
Cellulases produced by transgenic plants of the present invention can also be used in the textile, pulping, and paper-making industries. For instance, cellulases are conventionally used to treat denim fabrics to give them a xe2x80x9cstone-washedxe2x80x9d appearance. Cellulases are also used to modify paper pulps by digesting the cellulose fibers contained within the pulp. The cellulases produced by the transgenic plants described herein can be used in this fashion.